Thermodynamic and Structural Studies of Carbohydrate Binding by the Agrin-G3 Domain
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- 作者：Christine O. Sallum ; Richard A. Kammerer ; Andrei T. Alexandrescu
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：August 21, 2007
- 年：2007
- 卷：46
- 期：33
- 页码：9541 - 9550
- 全文大小：360K
- 年卷期：v.46,no.33(August 21, 2007)
- ISSN：1520-4995
文摘
Agrin is a key heparan sulfate proteoglycan involved in the development and maintenance ofsynaptic junctions between nerves and muscles. Agrin's important functions include clustering acetylcholinereceptors on the postsynaptic membranes of muscles and binding to the muscle protein 
-dystroglycanthrough its glycan chains. ITC and NMR were used to study the interactions of the C-terminal domain,agrin-G3, with carbohydrates implicated in agrin's functions. Sialic acid caps the glycan chains of-dystroglycan and occurs as a posttranslational modification on the muscle-specific kinase componentof the agrin receptor. We found that agrin-G3 binds sialic acid in a Ca2+-dependent manner. ITC dataindicate that binding is exothermic and occurs with a 1:1 stoichiometry. NMR chemical shift changesmap the sialic acid binding site to the loops that control the domain's acetylcholine receptor clusteringactivity. By contrast, the glycosaminoglycans heparin and heparan sulfate bind independently of Ca2+.Binding is endothermic, and the binding site spans about 12 saccharide units. The binding site for heparinoccupies a similar location but is distinct from that for sialic acid. NMR translational diffusion experimentsshow that agrin-G3 binds heparin with a 2:1 stoichiometry. Comparisons between the muscle (B0) andneuronal (B8) isoforms of the agrin domain showed very similar Ca2+ and carbohydrate binding properties.Our work identifies agrin-G3 as a functional analogue of the concanavalin A-type lectins, highlightsfunctional similarities between agrin and laminin G domains, and provides mechanistic clues about theroles of carbohydrates in agrin's functions.
-dystroglycanthrough its glycan chains. ITC and NMR were used to study the interactions of the C-terminal domain,agrin-G3, with carbohydrates implicated in agrin's functions. Sialic acid caps the glycan chains of-dystroglycan and occurs as a posttranslational modification on the muscle-specific kinase componentof the agrin receptor. We found that agrin-G3 binds sialic acid in a Ca2+-dependent manner. ITC dataindicate that binding is exothermic and occurs with a 1:1 stoichiometry. NMR chemical shift changesmap the sialic acid binding site to the loops that control the domain's acetylcholine receptor clusteringactivity. By contrast, the glycosaminoglycans heparin and heparan sulfate bind independently of Ca2+.Binding is endothermic, and the binding site spans about 12 saccharide units. The binding site for heparinoccupies a similar location but is distinct from that for sialic acid. NMR translational diffusion experimentsshow that agrin-G3 binds heparin with a 2:1 stoichiometry. Comparisons between the muscle (B0) andneuronal (B8) isoforms of the agrin domain showed very similar Ca2+ and carbohydrate binding properties.Our work identifies agrin-G3 as a functional analogue of the concanavalin A-type lectins, highlightsfunctional similarities between agrin and laminin G domains, and provides mechanistic clues about theroles of carbohydrates in agrin's functions.